Stress-responsive induction of a therapeutic agent and methods of use

ABSTRACT

This invention relates to compositions and methods for selective expression of a heterologous nucleic acid sequence in a targeted tissue, and more particularly to the glucose regulated protein 78 (grp78) stress-responsive promoter and its use in gene therapy and the production of transgenic animals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/141,505, filed Jun. 28, 1999, the disclosure of which isincorporated herein by reference.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

Pursuant to 35 U.S.C. '202(c), it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Nos. CA27607 and CA59318.

TECHNICAL FIELD

This invention relates to compositions and methods for selectiveexpression of a heterologous nucleic acid sequence in a targeted tissue,and more particularly to the glucose regulated protein 78 (grp78)stress-responsive promoter and its use in gene therapy and theproduction of transgenic animals.

BACKGROUND

Targeted gene expression is one of the most difficult and importantgoals in the development effective therapies for a variety of disorders,including, for example, cell proliferative disorders such as cancer orbiological stress resulting from glucose starvation in diseases such asdiabetes. Two strategies for specific expression include: 1) targetableentry; and 2) tissue or cell type specific gene expression. Targetableentry involves vector engineering to change vector binding tropism thusallowing cell type specific transduction. Tissue or cell specificexpression relies on restricting expression of the delivered geneexclusively to a particular type of tissue, such as a tumor.

Successful application of any method for targeting a specific tissue orcell for expression of a particular molecule (e.g., protein or nucleicacid) requires maximization of expression of the molecule in thetargeted environment. The most common promoter used to drive expressionof a foreign gene has been a constitutive, general-purpose viralpromoter such as the MuLV LTR. These promoters, while effective invitro, often fail to express the sequences under their control within abiologically stressed environment (Palmer et al., Proc. Natl. Acad. Sci.USA, 88:1330, 1991; Gazit et al., Cancer Res., 55:1660, 1995). Thesedata suggest that the MuLV promoter and other constitutive or cellularpromoters are not optimal for expressing a nucleic acid sequence within,for example, a fast growing solid tumor devoid of nutrients due toinsufficient blood supply. Further, even if a viral promoter escapesgenomic silencing, the expression pattern of the foreign gene will beconstitutive in normal as well as tumor cells. Such unregulatedexpression could be highly problematic in gene therapy methods.

To circumvent these difficulties, stress-responsive promoters provide anattractive means for tissue-specific expression of a therapeutic agent.For example, most fast growing tumors have a heterogeneous distributionof blood supply; by having a high interstitial and a low intravascularpressure, a decrease in nutrient supply results, leading to necrosis inthe center of the tumor. Glucose deprivation, calcium deprivation,chronic anoxia and low pH known to persist in poorly vascularized solidtumors induce a class of stress proteins referred to as theglucose-regulated proteins (GRPs) (Gazit et al., Cancer Res., 55:1660,1995; Koong et al., Int. J. Radiat. Oncol. Biol. Phys., 28:661, 1994)including the grp78 gene. A rat grp78 promoter has been used as a potentinternal promoter in a retroviral vector to drive expression of theneomycin phosphotransferase (neo) reporter gene in a murine fibrosarcomamodel system (Gazit et al., Cancer Res., 55:1660, 1995). Such a promoterprovides an attractive means for specifically expressing a therapeuticagent in a biologically stressed tissue using currently availablemethods in gene therapy.

There are several strategies that have been developed to accomplish genetherapy for the treatment of disorders that give rise to a biologicallystressed cellular environment, such as cancer or diabetes, for example.Within these strategies, there is a need for controlled, sustained,site-specific expression of a therapeutic agent such that surroundinghealthy tissue remains unaffected by the effects of the therapeuticagent.

SUMMARY

The present invention is based, in part, on the discovery that astress-responsive promoter specifically drives the expression of atherapeutic agent in vivo resulting in the efficient treatment of abiological stress-related disorder. Accordingly, in one embodiment, theinvention provides a nucleic acid construct comprising at least onestress-responsive non-coding regulatory sequence which comprises atleast two endoplasmic reticulum stress elements (ERSE) as set forth inSEQ ID NO:1, and a heterologous nucleic acid sequence operatively linkedto the regulatory sequence, wherein expression of the heterologoussequence is regulated by the non-coding sequence and wherein theheterologous sequence encodes a therapeutic agent effective for treatinga cell proliferative disorder.

In another aspect, the invention provides a nucleic acid constructcomprising at least one stress-responsive non-coding regulatory sequencewhich comprises at least two endoplasmic reticulum stress elements(ERSE) as set forth in SEQ ID NO:1; and a heterologous nucleic acidsequence operatively linked to the regulatory sequence, whereinexpression of the heterologous sequence is regulated by the non-codingsequence and wherein the heterologous sequence encodes a detectablemarker.

In one aspect, the present invention provides vectors comprising theaforementioned nucleic acid construct.

In another aspect, the present invention provides compositions usefulfor gene therapy, such as viral vectors comprising a nucleic acidconstruct of the invention.

The present invention also relates to the use of the before describednucleic acid construct and vectors for the preparation of pharmaceuticalcompositions for treating, preventing, and/or delaying a disease in asubject, such as, for example, a cell proliferative disease.Furthermore, the recombinant nucleic acid construct and vectors of theinvention can be used for the preparation of pharmaceutical compositionsfor identifying a tumorous disease in a human and non-human animal.

In a further embodiment, the present invention provides cells andtransgenic non-human animals, comprising the aforementioned recombinantnucleic acid sequence or vectors stably integrated into their genome andtheir use for the identification of substances capable of suppressing oractivating transcription from a stress-responsive regulatory sequence.

In a further embodiment, the invention provides a method of method forproducing a transgenic non-human animal having a phenotype characterizedby expression of a heterologous nucleic acid sequence encoding adetectable marker otherwise not naturally occurring in the animal,wherein the heterologous nucleic acid sequence is operably associatedwith at least one stress-responsive non-coding regulatory sequencecomprising at least two endoplasmic reticulum stress elements (ERSE) asset forth in SEQ ID NO:1, the method comprising: a) introducing at leastone transgene into a embryo of an animal, the transgene comprising atleast one stress-responsive non-coding regulatory sequence comprising atleast two endoplasmic reticulum stress elements (ERSE) as set forth inSEQ ID NO:1 isolated upstream from the heterologous nucleic acidsequence encoding a detectable marker; b) transplanting the embryo intoa pseudopregnant animal; c) allowing the embryo to develop to term; andd) identifying at least one transgenic offspring containing thetransgene.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows a schematic drawing of the recombinant retroviral vectors.In the GlNaGrpTk vector, the MuLV LTR drives the expression of neomycinphosphotransferase (neo) gene that is used as a selection marker. Inthis same vector, the grp78 promoter, (spanning nucleotides -520 to +175of the grp78 gene) drives the HSVtk gene. The grp78 promoter fragmentcontains three copies of the endoplasmic reticulum stress element(ERSE), the TATA box, and an internal ribosome entry site (IRES) in the5′ untranslated region downstream of its transcription initiation site(+1). In the GlTkSvNa vector, the MuLV LTR drives expression of theHSVtk gene, while the SV40 promoter drives the neo gene.

FIG. 2 shows induction of HSVTK by the grp78 promoter under glucosestarvation conditions. Panel A shows equal amounts of cell lysates fromthe parental B/C10ME cells, independently derived clonal cell linestransduced with G1TKSvNa (LTRtk#5), or transduced with GlNaGrpTk(grptk#1 and grptk#3) were subjected to Western blot analysis withantibodies against HSVTK, GRP78 and β-actin. The cells were grown undernormal culture medium (+) or glucose-starved (GS) conditions for 24 h.Panel B shows a bar graph indicating the intensity of the protein bandsquantitated by densitometry and normalized against that of actin servingas an internal loading control. The relative levels of HSVTK undernormal culture or glucose-starved conditions were plotted below theautoradiograms, with the protein level in control cells set as 1.

FIG. 3 shows the results of an in vitro GCV-sensitivity assay forB/C10ME cells. Panel A is a line graph showing about 5×10³ ofG1TkSvNa/clone #3 clones were seeded in duplicate into 6-well plates andincubated without (X) or with 0.1 (closed circles, open circles) μg/mlGCV starting at day 3 as indicated. The cells were then incubated innormal medium (-) or pretreated in glucose-free medium ( - - - ), andthe number of surviving cells were determined by the trypan blueexclusion method. Panel B shows data generated by the procedure used inA except that GlNaGrpTk/clone #3 cells were used. Panel C, in vitrobystander effect, non-transduced B/C10ME cells (TK) were co-culturedwith different ratio of B/C10ME clonal cell lines stably transfectedwith GlNaGrpTk. A total of 3,000 cells with various ratios were platedin quadruplicate in 96 well plate and treated with 10 mg/ml GCV for 10days. The number of remaining viable cells was measured by cellproliferation assay.

FIG. 4 shows tumor growth curves for B/C10ME fibrosarcoma. Panel A showsB/C10ME cells, B, three independently derived G1TkSvNa clonalderivatives (#2, #3, #5) or C, two independently derived GlNaGrpTkclonal derivatives (#1, #3) were used. Equivalent numbers of 2×10⁷viable cells were subcutaneously injected into BALB/c mice.Bi-perpendicular measurements were taken over a period of 29 days. GCV(as indicated by arrows) was administered daily starting at day 21 at adosage of 100 mg/kg of body weight.

FIG. 5 shows immunohistochemistry staining of HSVtk protein expressionin B/C10ME tumor tissues from mice. Panel A shows that, aftercounterstaining the tissue section with methyl green, no DAB stain canbe detected in tumor from non-transduced B/C10ME cells; B, isolatedpatches of HSVtk protein expression can be observed by cytoplasmic brownDAB staining in tumor from B/C10ME cells transduced with G1TkSvNa; andC, high level of HSVtk protein expression as shown by dark cytoplasmicbrown DAB staining in tumor from B/C10ME cells transduced withGlNaGrpTk. The magnification is 200×.

FIG. 6 shows micropet images of hypoxia inducible HSVtk expression in amurine mammary adenocarcinoma model. The mice were bearing tumorsderived from a murine mammary adenocarcinoma cell line, TSA, which hasbeen stably transfected with a retroviral vector, G1NaGRP-HSVtk,containing the GRP78 promoter that drives HSVtk gene expression.

FIG. 7 shows the presence of the LacZ transgene in transgenic mice.Panel A is a diagram of the grp78/LacZ Transgene construct comprisingabout 3000 base pairs of the grp78 regulatory sequence operably linkedto the LacZ gene. Panel B, upper gel, shows a Southern hybridizationresulting in the identification of a LacZ nucleic acid sequence intransgenic animals (Tg 132-147) containing the construct shown in PanelA. In the lower gel, a grp78 cDNA probe which hybridizes to the grp78gene was used to demonstrate that similar amounts of total DNA wereloaded in to each lane of the gel. The transgenic sequences wereidentified using a suitably labeled LacZ probe. Non-transgenic (Non-Tg)animals do not contain the LacZ sequence. Panel C is a bar graph showingthe LacZ activity present in hamster cells tranfected with a plasmidcontaining a nucleic acid construct shown in panel A (grp78/LacZ) or aplasmid expressing LacZ from the SV40 large T antigen promoter sequence(SV40/LacZ). Cells were treated with the calcium ionophore A23187 toinduce biological stress. Untreated and treated activity is indicated.

FIG. 8 shows a diagram of carcinogen treatment of wild-type (+/+),heterozygous for the grp78/LacZ transgene (Tg/+) or homozygous for thegrp78/LacZ transgene (Tg/Tg). The carcinogen (7,12-dimethylbenz[a]anthracene) was applied subcutaneously on a weekly basis over aperiod of six months. Subsequently, normal and tumorous tissue wereisolated and stained for detection of LacZ expression.

FIG. 9 shows color photographs of normal (non-neoplastic) tissue derivedfrom transgenic mice that are homozygous for the grp78/LacZ transgene(Tg/Tg) or tissue derived from wild-type (non-transgenic) mice (+/+).The mice were treated as described in FIG. 8.

FIG. 10 shows color photographs of tumorous tissues removed from micetreated as described in FIG. 8. Tissue from mice heterozygous for thegrp78/LacZ transgene (Tg/+), homozygous for the grp78/LacZ transgene(Tg/Tg) and wild-type (+/+) are indicated. Note that, followingLacZ-specific histological staining, LacZ expression is indicated intumorous tissue derived from Tg/+ mice as well as tissue derived fromTg/Tg mice.

FIG. 11 shows additional color photographs of tumorous tissues removedfrom mice treated as described in FIG. 8. Tissue from mice heterozygousfor the grp78/LacZ transgene (Tg/+) or homozygous for the grp78/LacZtransgene (Tg/Tg) are indicated. Note that, following LacZ-specifichistological staining, LacZ expression is indicated in tumorous tissuederived from Tg/+ mice as well as tissue derived from Tg/Tg mice.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The present invention is directed to compositions and methods fortreating a subject diagnosed as having a condition that can be treatedby gene therapy. The invention provides a means and method fordelivering at least one stress-responsive non-coding regulatory sequencecomprising at least two endoplasmic reticulum stress elements (ERSE) asset forth in SEQ ID NO:1; and a heterologous nucleic acid sequenceoperatively linked to the regulatory sequence, wherein expression of theheterologous sequence is regulated by the non-coding sequence. Thenon-coding regulatory sequence comprising the ERSE nucleic acidsequences can be derived, for example, from the transcription regulatorysequence of the glucose responsive protein 78 (grp78) gene. In addition,the invention provides transgenic animals the cells of which arehomozygous or heterozygous for the expression of a heterologous nucleicacid sequence driven by a stress-responsive promoter sequence. Suchanimals are useful, for example, for identifying glucose starved,calcium deprived or hypoxic tissue present in the animal duringdevelopment or upon exposure to mitogenic compounds, such ascarcinogens. Further, such animals can be used as models for thedevelopment of techniques for the identification of biologicallystressed tissue associated with, for example, cell proliferativedisorders, such as cancer or disorders associated with inflammation,such as arthritis.

The identification of endoplasmic reticulum stress elements (ERSE)allows for the development of a nucleic acid construct comprising astress-responsive regulatory sequence operably associated with aheterologous nucleic acid sequence. Such a construct can be incorporatedin, for example, a vector suitable for gene therapy. As used herein, aERSE nucleic acid sequence derived from a grp78 regulatory sequencemeans a nucleic acid sequence as set forth in SEQ ID NO:1. It isbelieved that the ERSE sequence of the invention can be incorporatedinto any non-coding regulatory sequence that provides appropriatetranscriptional and translational initiation regions for expression of aheterologous sequence in an animal cell. Preferably, a non-codingregulatory sequence comprising an ERSE nucleic acid sequence of theinvention is derived from the glucose responsive protein 78 (grp78)promoter sequence comprising a sequence from about 3000 base pairs 5′ ofthe site of initiation of transcription of the grp78 coding sequence toabout 200 base pairs 3′ of the site of initiation of the grp78 codingsequence, constituting a 3200 base pair regulatory region of the grp78gene.

A construct of the invention can be used in conjunction with aheterologous nucleic acid sequence encoding a therapeutic agent. Atherapeutic agent can encode a suicide gene for treating a cellproliferative disorder such as cancer or a therapeutic agent can encodea protein useful for ameliorating the adverse effects of glucosestarvation in the cell of a diabetic subject, for example. In addition,the present invention allows for the production of non-human transgenicanimals that express a heterologous nucleic acid sequence from a grp78regulatory sequence. This exemplary animal model provides a system foridentifying, for example, factors associated with tissue that isbiologically stressed, such as tumorous or inflammatory tissues. As usedherein, the term “biologically stressed” includes any cellularenvironment indicative of cellular distress, damage or trauma resultingin the activation of specific factors that respond to such anenvironment. For example, a biologically stressed tissue can result in acellular environment that is glucose starved, calcium deprived, hypoxic,acidic or in a pathological state. Biologically stressed furtherincludes tissue generating free radicals, or tissue that is hot or cold,inflamed or transformed or any other biological state indicative ofstressed tissue.

The grp78 gene regulatory sequence is located from about 3000 base pairs5′ of the site of initiation of transcription of the grp78 codingsequence to about 200 base pairs 3′ of the site of initiation of thegrp78 coding sequence and exhibits strong expression in biologicallystressed tissue, such as tissue that is glucose starved or hypoxic.Thus, a nucleic acid construct of the invention can include a 3200 basepair regulatory sequence derived from the grp78 gene. The genetic codefor endoplasmic reticulum stress signaling leading to grp gene inductionconsists of two units of a 19 base pair (bp) sequence motif(CCAAT)N9(CCACG) (SEQ ID NO:1) termed ERSE. This sequence contains atripartite structure, with a high affinity CBF/NF-Y binding siteseparated by precisely 9 by of a GC rich sequence motif to a lowaffinity YY1 binding site. The transcription regulatory sequencesfurther include transcriptional control regions such as TATAA and CAATbox sequences as well as sequences that regulate the tissue specificity(i.e., biologically stressed tissue) of the transcribed product. In thenucleic acid construct of the invention, the ATG start codon istypically provided by the nucleic acid sequence expressing the productof interest. As used herein, a “nucleic acid construct” of the inventionincludes at least one, or multiple, stress-responsive non-codingregulatory sequences and a heterologous nucleic acid sequenceoperatively linked to the regulatory sequence, wherein expression of theheterologous sequence is regulated by the non-coding sequence. A nucleicacid construct of the invention can be included in an expression vector.An “expression vector” refers to a plasmid, virus or other vehicle knownin the art that has been manipulated by insertion or incorporation ofthe nucleic acid construct of the invention. The expression vectortypically contains an origin of replication, as well as specific genesthat allow phenotypic selection of the transformed cells. Vectorssuitable for use in the present invention are well known in the art.

As used herein, the term “regulatory sequence” or “regulatory element”refers to a nucleic acid sequence capable of controlling thetranscription of an operably associated gene. A regulatory sequence ofthe invention may include a promoter, an enhancer and/or a silencer, forexample. Therefore, placing a gene under the regulatory control of apromoter or a regulatory element means positioning the gene such thatthe expression of the gene is controlled by the regulatory sequence(s).In general, promoters are found positioned 5′ (upstream) of the genesthat they control. Thus, in the construction of promoter genecombinations, the promoter is preferably positioned upstream of the geneand at a distance from the transcription start site that approximatesthe distance between the promoter and the gene it controls in thenatural setting. As is known in the art, some variation in this distancecan be tolerated without loss of promoter function. Similarly, thepreferred positioning of a regulatory element, such as an enhancer, withrespect to a heterologous nucleic acid sequence placed under its controlreflects its natural position relative to the structural gene itnaturally regulates. Enhancers are believed to be relatively positionand orientation independent in contrast to promoter elements. Thenoncoding sequences or intron sequences (e.g., which contain regulatorysequences) that are used in the invention construct are not more thanabout 9 kbp in length.

Regulatory sequence function during expression of a gene under itsregulatory control and can be tested at the transcriptional stage usingDNA/RNA and RNA/RNA hybridization assays (e.g., in situ hybridization,nucleic acid hybridization in solution or solid support) and at thetranslational stage using specific functional assays for the proteinsynthesized (e.g., by enzymatic activity, by immunoassay of the protein,by in vitro translation of mRNA or expression in microinjected xenopusoocytes).

As used herein, the term “nucleic acid sequence” refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. Nucleic acidsexpressing the products of interest can be assembled from cDNA fragmentsor from oligonucleotides which provide a synthetic gene which is capableof being expressed in a recombinant transcriptional unit. Polynucleotideor nucleic acid sequences of the invention include DNA, RNA and cDNAsequences.

Nucleic acid sequences utilized in the invention can be obtained byseveral methods. For example, the DNA can be isolated usinghybridization procedures that are well known in the art. These include,but are not limited to:

(1) hybridization of probes to genomic or cDNA libraries to detectshared nucleotide sequences; (2) antibody screening of expressionlibraries to detect shared structural features and (3) synthesis by thepolymerase chain reaction (PCR). Sequences for specific genes can alsobe found in GenBank, National Institutes of Health computer database.

The term “heterologous nucleic acid sequence” as used herein refers toat least one structural gene that is operably associated with theregulatory sequence of the invention. The nucleic acid sequenceoriginates in a foreign species, or, in the same species ifsubstantially modified from its original form. For example, the term“heterologous nucleic acid sequence” includes a nucleic acid originatingin the same species, where such sequence is operably linked to aregulatory sequence that differs from the natural or wild-typeregulatory sequence (e.g., grp78 regulatory sequence). Thus, anon-coding regulatory sequence of the invention can be operativelylinked to a heterologous nucleic acid sequence that is regulated by thenon-coding sequence.

The term “operably associated” refers to functional linkage between theregulatory sequence and the nucleic acid sequence regulated by theregulatory sequence. The operably linked regulatory sequence controlsthe expression of the product expressed by the nucleic acid sequence.Alternatively, the functional linkage also includes an enhancer element.

“Promoter” means the minimal nucleotide sequence sufficient to directtranscription. Also included in the invention are those promoterelements that are sufficient to render promoter-dependent nucleic acidsequence expression controllable for cell-type specific, tissuespecific, or inducible by external signals or agents; such elements maybe located in the 5′ or 3′ regions of the native gene, or in theintrons.

“Gene expression” or “nucleic acid sequence expression” means theprocess by which a nucleotide sequence undergoes successfultranscription and translation such that detectable levels of thedelivered nucleotide sequence are expressed in an amount and over a timeperiod so that a functional biological effect is achieved. “Expressiblegenetic construct” as used herein means a construct that has the grp78regulatory sequences positioned with a heterologous nucleic acidsequence encoding a desired product, such that the nucleic acid sequenceis expressed.

A heterologous nucleic acid sequence of the invention can encode a“therapeutic agent” effective for treating, for example, a cellproliferative disorder or a disorder associated with glucose starvation,such as diabetes. As used herein, a “therapeutic agent” can include astructural gene that encodes a biologically active protein of interest.The term “structural gene” excludes the non-coding regulatory sequencethat drives transcription. The structural gene may be derived in wholeor in part from any source known to the art, including a plant, afungus, an animal, a bacterial genome or episome, eukaryotic, nuclear orplasmid DNA, cDNA, viral DNA or chemically synthesized DNA. A structuralgene may contain one or more modifications in either the coding or theuntranslated regions which could affect the biological activity or thechemical structure of the expression product, the rate of expression orthe manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions and substitutions ofone or more nucleotides. The structural gene may constitute anuninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. The structural gene may alsoencode a fusion protein. It is contemplated that introduction intoanimal tissue of nucleic acid constructs of the invention will includeconstructions wherein the structural gene and its regulatory sequenceare each derived from different animal species.

A structural gene can encode an enzyme, such as a drug-metabolizingenzyme that confers a dominant, negatively selectable phenotype to acell, such as cell death. Such a gene can encode an enzyme that canconvert a non-therapeutically effective compound in to a therapeuticallyeffective compound. For example, the activation of a relatively nontoxic(i.e., non-therapeutically effective) prodrug to a cytotoxic (i.e.,therapeutically effective) compound in a specifically targeted tissuecan be used to effectively treat a cell proliferative disorder. Enzymescapable of performing such a function include herpes simplex virus (HSV)thymidine kinase, vesicular stomatitis virus (VSV) thymidine kinase,deoxycytidine kinase, cytosine deaminase or nucleoside phosphorylase.Prodrugs converted by the aforementioned enzymes include ganciclovir,acyclovir, 6-methoxypurine arabinoside (Ara-M), cytosine arabinoside orcytarabine (Ara-C), fludarabine, 2-chlorodeoxyadenosine,difluorodeoxycytidine, 5-fluorocytidine and6-methylpurine-2′-deoxyriboside (MeP-dr).

Because current gene transfer techniques are unable to achieve asatisfactorily high level of transfer efficiency in an in vivo setting,alternative strategies that do not require 100% efficiency of genetransfer have been sought. Two general approaches have evolved that maybe effective when only a minority of the tumor cells are transduced: (1)cell-targeted suicide, achieved by directing the synthesis of a toxicmetabolite that can permeate the tumor microenvironment, and (2)engineering an immune response to the tumor cells by ectopic cytokineexpression or other means for immune recognition or activation.

Examples of genes encoding therapeutic agents that can be used in theinvention construct include genes encoding enzymes that convert aprodrug to a toxic metabolite. As noted above, a variety of enzymes arecapable of performing such a function, and typically kill cells byactivation of a relatively nontoxic prodrug to a cytotoxic form. Greaterselectivity in killing malignant cells will be obtained if thetransferred gene is not normally found in human beings (e.g.,HSV-thymidine kinase), rather than by overexpressing an endogenous gene(e.g., deoxycytidine kinase).

The tumoricidal activity of the HSV-TK/ganciclovir system is due toseveral factors. In dividing cells, the phosphorylated ganciclovirinhibits DNA synthesis. This effect is not confined to cells that aredirectly transduced with HSV-TK, as neighboring cells are also affected.This phenomenon, which likely occurs as a result of several mechanisms,has been termed the “bystander effect” and has been observed in severaltumor types, including CNS tumors. Transfer of the phosphorylatedganciclovir between cells (“metabolic cooperation”) via gap junctionshas been proposed as a possible mechanism. Phagocytosis by neighboringcells of ganciclovir phosphate-containing apoptotic vesicles (from dyingtransduced cells) also has been proposed.

In addition, a therapeutic agent of the invention includes nucleic acidsequences encoding tumor suppressor proteins such as p53 (Takahashi etal. Cancer Res. 62:2340, 1992) and Retinoblastoma (RB); and nucleic acidsequences encoding apoptosis or cell death promoting proteins such asFas (Itoh et al., Cell 66:233, 1991), GAX (PCT/US95/01882), and FADD(Chinnalyan et al. Cell, 81:505, 1995) which interacts with the deathdomain of Fas and initiates apoptosis.

A therapeutic agent of the invention also includes nucleic acidsequences that encode cell cycle blockers such as GATA-6 (Suzuki et al,Genomics, 38:283, 1996), anti-angiogenesis proteins such as endostatinand angistatin (Folkman J., Nature Med. 1:27, 1995), anti-sense genesequences (Wang, Nature Med. 3:887, 1997), and viral subunit vaccines(Donnelly et al. Nature Med. 1:583, 1995).

A therapeutic agent also encompasses those sequences encoding proteins,such as asparaginase, that induce cell death by depriving a cell of anecessary metabolite. Asparaginase induces apoptosis by catalyzing thehydrolysis of circulating asparagine to aspartic acid and ammonia, thusdepriving cells of the asparagine necessary for protein synthesis,leading to cell death.

A therapeutic agent of the invention also includes immunomodulators andother biological response modifiers. The term “biological responsemodifiers” encompasses substances that are involved in modifying theimmune response in such manner as to enhance the destruction of tumor,for example. Examples of immune response modifiers include suchcompounds as lymphokines. Lymphokines include tumor necrosis factor, theinterleukins, lymphotoxin, macrophage-activating factor, migrationinhibition factor, colony stimulating factor, and interferon. Includedin this category are immunopotentiating agents including nucleic acidsencoding a number of the cytokines classified as “interleukins”. Theseinclude, for example, interleukins 1 through 12. Also included in thiscategory, although not necessarily working according to the samemechanisms, are interferons, and in particular gamma interferon (γ-IFN),tumor necrosis factor (TNF) and granulocyte-macrophage-colonystimulating factor (GM-CSF). Nucleic acids encoding growth factors,toxic peptides, ligands, receptors, suicide factors (e.g., TK) or otherphysiologically important proteins can also be introduced into specificcells of the prostate.

Further, a therapeutic agent includes sense or antisense nucleic acidsencoded by a heterogenous nucleic acid of the invention. For example, asense polynucleotide sequence (the DNA coding strand) encoding apolypeptide can be introduced into the cell to increase expression of a“normal” gene. Other cell disorders can also be treated with nucleicacid sequences that interfere with expression at the translationallevel. This approach utilizes, for example, antisense nucleic acid,ribozymes, or triplex agents to block transcription or translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or triplex agent, or by cleaving it with a ribozyme. Alternatively,the method includes administration of a reagent that mimics the actionor effect of a gene product or blocks the action of the gene. Therefore,when a cell proliferative disorder, such as cancer, is etiologicallylinked with over expression of a polynucleotide, it would be desirableto administer an inhibiting reagent such as an antisense polynucleotide.For example, overexpression of the bcl-2 gene that is translocated innodular non-Hodgkin's lymphomas, inactivates a key pathway of programmedcell death (apoptosis) and leads to continuous proliferation andsurvival of highly mutated tumor cells that have the capacity to surviveDNA damage. Similarly, an increase in expression of the D cyclin (theprad oncogene) promotes cell entry into DNA synthesis. Additionaloncogenes that promote cell proliferation include ABL, ERBB-1, ERBB-2(NEU), GIP, GSP, MYC, L-MYC, N-MYC, H-RAS, RET, ROS, K-SAM, SIS, SRC,C-FOS, C-JUN AND TRK. Thus, efforts directed toward restoring apoptosisin tumor cells, by inhibiting the overexpression of an apoptosisinhibitor, such as bcl-2, or cell proliferation promoting oncogene, suchas Ras, can be accomplished using antisense methodology.

The use of antisense methods to inhibit the in vitro translation ofgenes is well known in the art (see, e.g., Marcus-Sakura, Anal.Biochem., 172:289, 1988). Antisense nucleic acids are nucleic acidmolecules (e.g., molecules containing DNA nucleotides, RNA nucleotides,or modifications (e.g., modification that increase the stability of themolecule, such as 2′-O-alkyl (e.g., methyl) substituted nucleotides) orcombinations thereof) that are complementary to, or that hybridize to,at least a portion of a specific nucleic acid molecule, such as an RNAmolecule (e.g., an mRNA molecule) (see, e.g., Weintraub, ScientificAmerican, 262:40, 1990). The antisense nucleic acids hybridize tocorresponding nucleic acids, such as mRNAs, to form a double-strandedmolecule, which interferes with translation of the mRNA, as the cellwill not translate a double-stranded mRNA. Antisense nucleic acids usedin the invention are typically at least 10-12 nucleotides in length, forexample, at least 15, 20, 25, 50, 75, or 100 nucleotides in length. Theantisense nucleic acid can also be as long as the target nucleic acidwith which it is intended to form an inhibitory duplex. As is describedfurther below, the antisense nucleic acids can be introduced into cellsas antisense oligonucleotides, or can be produced in a cell in which anucleic acid encoding the antisense nucleic acid has been introduced by,for example, using gene therapy methods.

Gene Therapy

The present invention also provides gene therapy for the treatment of acell proliferative disorder. Such therapy would achieve its therapeuticeffect by introduction of the nucleic acid construct of the inventioninto cells having the disorder such that a heterologous nucleic acidsequence encoding a therapeutic agent or a detectable marker isexpressed from a stress-responsive non-coding regulatory sequence.Preferably, the regulatory sequence is isolated from a glucoseresponsive protein 78 (grp78), however any regulatory sequence suitablefor expression biologically stressed cells and/or tissue can be used inthe present invention.

Delivery of a nucleic acid construct of the invention can be achieved byintroducing the construct into a cell using a variety of methods knownto those of skill in the art. For example, the construct can bedelivered into a cell using a colloidal dispersion system.Alternatively, nucleic acid construct of the invention can beincorporated (i.e., cloned) into an appropriate vector. For example, arecombinant vector of the invention can be an expression vector suitablefor expression of the heterologous sequence in a target cell, such as acell that is biologically stressed. Preferably, a recombinant vectorcomprising nucleic acid construct of the invention includes areplication competent or replication incompetent recombinant viralvector. For example, a recombinant viral vector of the invention can bederived from an RNA virus (i.e., retrovirus) such s lentivirus, or a DNAvirus such as adenovirus. Delivery of a construct of the invention intoa cell can be performed in vivo or ex vivo. Further, methods of theinvention can be performed alone or in conjunction with standard medicaltreatments currently available for treating a cell proliferativedisorder. For example, when a tumor is being treated, it may bepreferable to remove the majority of a tumor surgically or by radiationprior to introducing a construct of the invention in to the cellscomprising the tumor.

Various viral vectors that can be utilized for gene therapy, as taughtherein, include DNA viruses such as adenovirus, herpes virus, vaccinia,or an RNA virus such as a retrovirus. The retroviral vector can be aderivative of a retrovirus capable of infecting a mammalian host cell.Examples of retroviral vectors in which a foreign gene can be insertedinclude, but are not limited to: Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when the subject is ahuman, a vector such as the gibbon ape leukemia virus (GaLV) isutilized. A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate anucleic acid construct of the invention into a target cell. By insertingthe construct of the invention into the viral vector along with anothergene that encodes ligand for a receptor on a specific target cell, forexample, the vector is now target cell entry specific as well targetcell expression specific. Preferred targeting is accomplished by usingan antibody to target the retroviral vector. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome, for example, to allow target specific delivery of theretroviral vector containing the construct of the invention.

Retroviruses are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The family Retroviridae areenveloped single-stranded RNA viruses that typically infect mammals aswell as avian species. Retroviruses are unique among RNA viruses in thattheir multiplication involves the synthesis of a DNA copy of the RNAthat is then integrated into the genome of the infected cell.

The Retroviridae family consists of three groups: the spumaviruses (orfoamy viruses) such as the human foamy virus (HFV); the lentiviruses, aswell as visna virus of sheep; and the oncoviruses (although not allviruses within this group are oncogenic). The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV).

Retroviruses are defined by the way in which they replicate theirgenetic material. During replication the RNA is converted into DNA.Following infection of the cell a double-stranded molecule of DNA isgenerated from the two molecules of RNA that are carried in the viralparticle by the molecular process known as reverse transcription. TheDNA form becomes covalently integrated in the host cell genome as aprovirus, from which viral RNAs are expressed with the aid of cellularand/or viral factors. The expressed viral RNAs are packaged intoparticles and released as infectious virion.

Retroviruses can be transmitted horizontally and vertically. Efficientinfectious transmission of retroviruses requires the expression on thetarget cell of receptors that specifically recognize the viral envelopeproteins, although viruses may use receptor-independent, nonspecificroutes of entry at low efficiency. In addition, the target cell typemust be able to support all stages of the replication cycle after virushas bound and penetrated. Vertical transmission occurs when the viralgenome becomes integrated in the germ line of the host. The proviruswill then be passed from generation to generation as though it were acellular gene. Hence endogenous proviruses become established whichfrequently lie latent, but which can become activated when the host isexposed to appropriate agents.

Numerous gene therapy methods that take advantage of retroviral vectorsfor treating a wide variety of diseases are known in the art (see, e.g.,U.S. Pat. Nos. 4,405,712 and 4,650,764; Friedmann, 1989, Science,244:1275-1281; Mulligan, 1993, Science, 260:926-932, R. Crystal, 1995,Science 270:404-410, each of which are incorporated herein by referencein their entirety). An increasing number of these methods are currentlybeing applied in human clinical trials (Morgan, 1993, BioPharm,6(1):32-35; see also The Development of Human Gene Therapy, TheodoreFriedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1999. ISBN 0-87969-528-5, which is incorporated herein byreference in its entirety). The safety of these currently available genetherapy protocols can be substantially increased by using retroviralvectors of the present invention. For example, where the retroviralvector infects a non-targeted cell, the retroviral genome will integratebut the heterologous nucleic acid sequence will not be transcribedunless the cell or tissue is biologically stressed. However, when theretroviral vector containing a nucleic acid construct of the inventioninfects a targeted cell (i.e., a cell that is glucose starved, calciumdeprived, hypoxic, etc.) the activation of the stress-responsiveregulatory sequence will result in transcription and translation of theheterologous nucleic acid sequence.

Recombinant retroviruses defective for replication require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retro virus under thecontrol of regulatory sequences within the LTR. These plasmids aremissing a nucleotide sequence that enables the packaging mechanism torecognize an RNA transcript for encapsidation. Helper cell lines whichhave deletions of the packaging signal include but are not limited toΨ2, PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Another targeted delivery system useful for introducing a nucleicconstruct of the invention into a target cell is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles that are useful as delivery vehicles invitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 um can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,Trends Biochem. Sci., 6:77, 1981). In order for a liposome to be anefficient gene transfer vehicle, the following characteristics should bepresent: (1) encapsulation of the nucleic acid of interest (i.e., anucleic acid construct of the invention or a vector comprising theconstruct) at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

In preferred embodiments, the grp78 regulatory sequence comprises atleast one stress-responsive nucleic acid sequence regulatable by factorspresent in biologically stressed cells and tissues such as glucosestarved or hypoxic cells or tissue. In one aspect of the invention, theexpression of a heterologous nucleic acid sequence encoding atherapeutic agent or detectable marker is regulated by fusion of theheterologous nucleic acid, or a fragment thereof, to at least onestress-responsive regulatory sequence, such as, for example, a grp78regulatory sequence. A grp78 regulatory sequence is one that is notnormally associated with, and does not normally regulate, the expressionof a heterologous nucleic acid that it regulates in the practice of theinvention. Grp78 regulatory elements can comprise transcriptional,post-transcriptional, translational, and post-translational elements; aswell as regulatory elements related to replication. By way of example,grp78 transcriptional regulatory elements can include promoters,enhancers, operators, and elements that modulate the rate oftranscription initiation, elongation and/or termination;post-transcriptional regulatory elements can include those influencingmessenger stability, processing and transport; translational regulatoryelements can include those which modulate the frequency of translationinitiation and the rate of translational elongation; post-translationalregulatory elements can include those which influence proteinprocessing, stability and transport; and replication-associatedregulatory elements can include those related to gene dosage.

In one embodiment, the invention provides recombinant vectors comprisinga nucleic acid construct of the invention. The recombinant vectors aremade using standard methods of molecular biology and biotechnology toincorporate a nucleic acid construct of the invention containing aheterologous nucleic acid sequence in operative linkage with astress-responsive regulatory sequence, such as a grp-78 regulatorysequence. In preferred embodiments, the grp-78 regulatory sequence willbe upstream of the heterologous sequence when they are placed inoperative linkage. Locations of restriction enzyme recognition sequencescan be easily determined by one of skill in the art. Alternatively,various in vitro techniques can be used for insertion of a restrictionenzyme recognition sequence at a particular site, or for insertion ofnucleic acid construct at a site that does not contain a restrictionenzyme recognition sequence. Such methods include, but are not limitedto, oligonucleotide-mediated heteroduplex formation for insertion of oneor more restriction enzyme recognition sequences (see, for example,Zoller et al. (1982) Nucleic Acids Res. 10:6487-6500; Brennan et al.(1990) Roux's Arch. Dev. Biol. 199:89-96; and Kunkel et al. (1987) Meth.Enzymology 154:367-382) and PCR-mediated methods for insertion of longersequences. See, for example, Zheng et al. (1994) Virus Research31:163-186.

Operative linkage refers to an arrangement of one or more regulatorysequences with one or more coding sequences, such that the regulatorysequence(s) is capable of exerting its regulatory effect on the codingsequence.

By way of illustration, a stress responsive-transcriptional regulatorysequence or a promoter is operably linked to a heterologous sequence ifthe transcriptional regulatory sequence or promoter promotestranscription of the heterologous sequence. Similarly, an operator isconsidered operatively linked to a promoter or to a heterologoussequence if binding of a repressor to the operator inhibits initiationat the promoter so as to prevent or diminish expression of theheterologous sequence. An operably linked transcriptional regulatorysequence is generally joined in cis with the coding sequence, but it isnot necessarily directly adjacent to it.

Recombinant vectors comprising a nucleic acid construct of the inventioncan also comprise other types of sequence including, but not limited to,replication origins, detectable markers (including, but not limited to,those encoding antibiotic resistance), transcription termination sites,sequences specifying translation initiation and termination, sequencesmediating mRNA processing and/or stability and multiple cloning sites.

Recombinant vectors can exist as freely-replicating extrachromosomalelements, such as plasmids or episomes, or can exist as chromosomalrecombinants, such as would be achieved either by integration of anucleic acid construct into the chromosome of a cell. Methods forobtaining chromosomal integration of recombinant vectors have beendescribed, for example, by Gerhardt et al., METHODS FOR GENERAL ANDMOLECULAR MICROBIOLOGY, American Society for Microbiology, Washington,D.C., 1994; Link et al. (1997) J. Bacteriol. 179:6228-6237; and Metcalfet al. (1996) Plasmid 35:1-13.

A coding sequence, as present in a recombinant construct, can encode afull-length nucleic product (i.e., the length normally found in thewild-type cell) or any fragment of a gene product. A gene product can beRNA or a polypeptide; untranslated RNA gene products can includestructural, catalytic and regulatory RNA molecules. Examples ofuntranslated RNA gene products include, but are not limited to, tRNA,rRNA, antisense RNAs and ribozymes. In one embodiment, a coding sequencecomprises a gene, which can encode a therapeutic agent, or a geneproduct whose function is to act as a detectable marker under aparticular set of environmental conditions. It is understood that anygene of interest can be placed in operative linkage with grp-78regulatory region sequences, so that its expression is regulated by thegrp78 regulatory region sequences.

The phrase “non-dividing” cell refers to a cell that does not go throughmitosis. Non-dividing cells may be blocked at any point in the cellcycle, (e.g., G0/G1, G1/S, G2/M), as long as the cell is not activelydividing. For ex vivo infection, a dividing cell can be treated to blockcell division by standard techniques used by those of skill in the art,including, irradiation, aphidocolin treatment, serum starvation, andcontact inhibition. However, it should be understood that ex vivoinfection is often performed without blocking the cells since many cellsare already arrested (e.g., stem cells). For example, a recombinantlentivirus vector of the invention is capable of infecting anynon-dividing cell, regardless of the mechanism used to block celldivision or the point in the cell cycle at which the cell is blocked.Examples of pre-existing non-dividing cells in the body includeneuronal, muscle, liver, skin, heart, lung, and bone marrow cells, andtheir derivatives. For dividing cells onco-retroviral vectors can beused.

By “dividing” cell is meant a cell that undergoes active mitosis, ormeiosis. Such dividing cells include stem cells, skin cells (e.g.,fibroblasts and keratinocytes), gametes, and other dividing cells knownin the art. Of particular interest and encompassed by the term dividingcell are cells having cell proliferative disorders, such as neoplasticcells. The term “cell proliferative disorder” refers to a conditioncharacterized by an abnormal number of cells. The condition can includeboth hypertrophic (the continual multiplication of cells resulting in anovergrowth of a cell population within a tissue) and hypotrophic (a lackor deficiency of cells within a tissue) cell growth or an excessiveinflux or migration of cells into an area of a body. The cellpopulations are not necessarily transformed, tumorigenic or malignantcells, but can include normal cells as well.

The present invention provides gene therapy for the treatment of cellproliferative disorders or disorders associated with glucose starvationsuch as diabetes. Such therapy would achieve its therapeutic effect byintroduction of a nucleic acid construct encoding an appropriatetherapeutic agent (e.g., suicide gene, tumor suppressor genes,antisense, ribozymes), into cells of subject having the disorder.Delivery of such a nucleic acid constructs can be achieved using a viralvector of the present invention.

Cell proliferative disorders include disorders associated with anovergrowth of connective tissues, such as various fibrotic conditions,including scleroderma, arthritis and liver cirrhosis. Cell proliferativedisorders include neoplastic disorders such as head and neck carcinomas.Head and neck carcinomas would include, for example, carcinoma of themouth, esophagus, throat, larynx, thyroid gland, tongue, lips, salivaryglands, nose, paranasal sinuses, nasopharynx, superior nasal vault andsinus tumors, esthesioneuroblastoma, squamous call cancer, malignantmelanoma, sinonasal undifferentiated carcinoma (SNUC) or bloodneoplasia. Also included are carcinoma's of the regional lymph nodesincluding cervical lymph nodes, prelaryngeal lymph nodes, pulmonaryjuxtaesophageal lymph nodes and submandibular lymph nodes (Harrison'sPrinciples of Internal Medicine (eds., Isselbacher, et al., McGraw-Hill,Inc., 13th Edition, pp 1850-1853, 1994). Other cancer types, include,but are not limited to, lung cancer, colon-rectum cancer, breast cancer,prostate cancer, urinary tract cancer, uterine cancer lymphoma, oralcancer, pancreatic cancer, leukemia, melanoma, stomach cancer andovarian cancer.

Disorders associated with glucose starvation include diabetes or anyother disorder wherein tissue is constantly or periodically subjected tolow glucose availability such that the cells of the tissue arebiologically stressed.

In addition, the therapeutic methods (e.g., the gene therapy or genedelivery methods) as described herein can be performed in vivo or exvivo. It may be preferable to remove the majority of a tumor prior togene therapy, for example surgically or by radiation.

The invention also provides a method of nucleic acid transfer to atarget cell to provide expression of a particular nucleic acid sequence(e.g., a heterologous sequence). Therefore, in another embodiment, theinvention provides a method for introduction and expression of aheterologous nucleic acid sequence in a target cell comprising infectingthe target cell with a recombinant virus of the invention containing anucleic acid construct of the invention and expressing the heterologousnucleic acid sequence in the target cell. As mentioned above, the targetcell can be any cell type including dividing, non-dividing, neoplastic,immortalized, modified and other cell types recognized by those of skillin the art, so long as they are capable of infection by a retrovirus.

In another embodiment, the invention provides a method of treating asubject having a cell proliferative disorder. The subject can be anymammal, and is preferably a human. The subject is contacted with arecombinant vector of the present invention. The recombinant vector ispreferably a recombinant viral vector and more preferably a recombinantretroviral vector. The contacting can be in vivo or ex vivo. Methods ofadministering the vector of the invention are known in the art andinclude, for example, systemic administration, topical administration,intraperitoneal administration, intra-muscular administration, as wellas administration directly at the site of a tumor or cell-proliferativedisorder and other routes of administration known in the art.

Pharmaceutical Compositions

The invention further includes various pharmaceutical compositionsuseful for treating a cell proliferative disorder or a disorderassociated with glucose starvation such as, for example, diabetes. Thepresent invention provides a nucleic acid construct capable of drivingthe expression of a therapeutic agent in a cell associated withbiologically stressed tissue.

A biologically stressed tissue of the invention includes those tissueswhere the cellular environment is “naturally” glucose starved, calciumdeprived, hypoxic, acidic or in a pathological state. Biologicallystressed further includes tissue generating free radicals, or tissuethat is hot or cold, inflamed or transformed or any other biologicalstate indicative of stressed tissue. A naturally biologically stressedtissue is a tissue wherein normal cellular metabolism in conjunctionwith a pathological state has induced the biological stress. Forexample, a fast growing solid tumor devoid of nutrients due toinsufficient blood supply exposes the neoplastic cells contained in suchan environment to glucose deprivation, calcium deprivation, chronicanoxia and low pH. Thus, the cells in such an environment are subjectedto biological stress that is induced by a pathological state resultingfrom tumor growth. The nucleic acid construct of the invention can beused to express a therapeutic agent, such as, for example, a suicidegene, or an apoptosis-inducing gene, such that the targeted cell iskilled. The surrounding healthy tissue remains unaffected by thetreatment because they do not provide a biologically stressed necessaryfor expression of the therapeutic agent.

In addition, diabetes is a disease that results in glucose starvation ina wide range of tissues. Cells subjected to glucose deprivation mustutilize other sources of energy in order to survive. The consequences ofthis type metabolism is a cellular environment that is, for example,acidic. Again, a nucleic acid construct of the invention can be used toexpress a therapeutic agent such that the acidic environment of atargeted cell can be ameliorated by expression of the agent.

A biologically stressed tissue of the invention also includes thosetissues where a biologically stressed cellular environment has been“artificially” induced. For example, photodynamic therapy involves thecombined use of photosensitizing drugs and light for the treatment ofmalignant or benign disease. The photosensitized chemical reactionrequires oxygen. Light, delivered to the tissue, activates porphyrinmolecules. These molecules transfer their energy to form cytotoxicsinglet oxygen, which results in lethal alteration of cellular membranesand subsequent tissue destruction. Artificial means for inducingbiological stress also include compounds such as combretastatinA4-phosphate (CA4DP). CA4DP has been used as an antiangiogenesis agentto prevent or reduce the blood supply to, for example, tumorous tissue.Reduced blood supply facilitated by CA4DP, or any other antiangiogenicagent, promotes biological stress in the affected tissue and providesthe appropriate environment for expression of a therapeutic agent of theinvention.

Thus, a nucleic acid construct of the invention can be used inconjunction with a method for “artificially” inducing a biologicallystressed cellular environment. For example, the construct can beintroduced into a cell as part of a pharmaceutical compositioncomprising, for example, a liposomal delivery vehicle or a viraldelivery vehicle, prior to, during, or subsequent to artificialinduction of biological stress. Potential uses in dermatology includethe treatment of malignant cutaneous lesions and nononcologicconditions, including psoriasis, alopecia, viral infections, andvascular malformations. Photodynamic therapy also has been employed forbladder, endobronchial, and esophageal carcinoma.

The pharmaceutical compositions according to the invention are preparedby placing a nucleic acid construct of the invention into a formsuitable for administration to a subject using carriers, excipients andadditives or auxiliaries. The nucleic acid construct can be contained ina recombinant vector, preferably a recombinant viral vector and mostpreferably a recombinant retroviral vector. A pharmaceutical compositioncan include a nucleic acid construct of the invention comprising atleast one stress-responsive non-coding regulatory sequence comprising atleast two endoplasmic reticulum stress elements (ERSE). Preferably, thestress-responsive non-coding regulatory sequence is derived from aglucose responsive protein 78 (grp78) gene. A heterologous nucleic acidsequence operatively linked to the regulatory sequence. The expressionof the heterologous sequence is regulated by the non-coding sequence andthe heterologous sequence can encode a therapeutic agent effective fortreating, for example, a cell proliferative disorder.

Generally, the terms “treating”, “treatment”, and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be therapeutic in terms of a partial or completecure for a cell proliferative disorder. “Treating” as used herein coversany treatment of (e.g., complete or partial), or prevention of, a cellproliferation disorder or for ameliorating the pathogenic effect ofbiological stress, such as biological stress induced by glucosedeprivation, in a mammal, particularly a human, and includes:

-   -   (a) preventing the disease from occurring in a subject that may        be predisposed to the disease, but has not yet been diagnosed as        having it;    -   (b) inhibiting the disorder, i.e., arresting the development of,        for example, a tumor; or    -   (c) relieving or ameliorating the disorder or disease, i.e.,        cause regression of the disorder or disease.

Thus, the invention includes various pharmaceutical compositions usefulfor ameliorating symptoms attributable to a cell proliferative disorderor, alternatively, for inducing a protective immune response to treat acell proliferative disorder or for ameliorating the pathogenic effect ofbiological stress. For example, a pharmaceutical composition accordingto the invention can be prepared to include a nucleic acid constructaccording to the invention into a form suitable for administration to asubject using carriers, excipients and additives or auxiliaries.Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975) and The National Formulary XIV., 14th ed. Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Langer, Science, 249:1527, (1990); Gilman et al. (eds.) (1990), each of which is hereinincorporated by reference.

As used herein, “administering a therapeutically effective amount” isintended to include methods of giving or applying a pharmaceuticalcomposition of the invention to a subject that allow the composition toperform its intended therapeutic function. The therapeutically effectiveamounts will vary according to factors such as the degree of infectionin a subject, the age, sex, and weight of the individual. Dosage regimacan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses can be administered daily or the dose canbe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The pharmaceutical composition can be administered in a convenientmanner such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example,with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the pharmaceutical composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5 toabout 80% of the weight of the unit.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the pharmaceuticalcomposition can be incorporated into sustained-release preparations andformulations.

As used herein, a “pharmaceutically acceptable carrier” is intended toinclude solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the pharmaceutical composition, usethereof in the therapeutic compositions and methods of treatment iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the pharmaceuticalcomposition and the particular therapeutic effect to be achieve, and (b)the limitations inherent in the art of compounding such anpharmaceutical composition for the treatment of a pathogenic infectionin a subject.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

Transgenic Animal Production

Transgenesis is a term used to describe the artificial introduction ofnew genetic material into the germ line of an organism. As such, it is aform of genetic manipulation that includes not only the introduction offoreign DNA into the germ line but also designer gene modificationswhich to date usually involve the insertion of new extraneous DNA.Transgenic animals are useful as models for diseases for the testing ofpharmacological agents prior to clinical trials or the testing oftherapeutic modalities.

Thus, in another embodiment, the present invention provides a transgenicnon-human animal containing a nucleic acid construct of the invention.As previously noted, a “nucleic acid construct” of the inventionincludes at least one, or multiple, stress-responsive non-codingregulatory sequences and a heterologous nucleic acid sequenceoperatively linked to the regulatory sequence, wherein expression of theheterologous sequence is regulated by the non-coding sequence. Thus, a“transgene”, as used herein, refers to a nucleic acid construct of theinvention that is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from that cell. For example, atransgene of the present invention can contain multiple grp78 regulatoryelements driving expression of a heterologous nucleic acid sequence inbiologically stressed tissue, such as glucose starved or hypoxic tissue.

Phenotypically, a transgenic animal of the present invention can appearnormal because of the unique stress-responsive regulatory sequence usedto develop the animal. Such a promoter is fully active only in acellular environment that exhibits the biochemical manifestations ofbiological stress. As previously noted, such a cellular environment caninclude, but is not limited to, glucose starvation, calcium deprivationor hypoxia. Thus, a transgene of the present invention may not be activeunder normal cellular conditions. However, when an animal having such atransgene incorporated in to its genome is exposed to conditions thatinduce biological stress in the whole animal or in specific tissues, thetransgene can become activated in the whole animal or only in specifictissues. For example, exposure of a transgenic animal of the inventionto a mitogenic agent can induce a cell proliferative disorder such thata tumor develops as a result of the exposure. As previously noted, afast growing solid tumor devoid of nutrients due to insufficient bloodsupply exposes the neoplastic cells contained in such an environment toglucose deprivation, calcium deprivation, chronic anoxia and low pH.Thus, a transgene containing a stress-responsive regulatory sequence,such as grp78, can become active in this environment.

The present invention provides transgenic animals that are heterozygousfor the transgene and animals that are homozygous for the transgene ofthe invention. As shown in FIGS. 10 and 11, both heterozygous andhomozygous animals display activity of the stress responsive regulatorysequence in tissues that have developed tumors, i.e. are biologicallystressed. Thus, it is understood that both heterozygous and homozygoustransgenic animals of the invention are useful, for example, foridentifying compounds that induce biological stress in such an animal.

A transgene of the invention includes a nucleic acid constructcomprising at least one stress-responsive regulatory sequence operablyassociated with a heterologous nucleic acid sequence. A heterologousnucleic acid sequence can encode a detectable marker expressed under thecontrol of a stress-responsive regulatory sequence that is active in atargeted, biologically stressed, tissue. For example, grp78 regulatorysequences can be used in conjunction with a heterologous nucleic acidsequence encoding a visually detectable marker, such as greenfluorescent protein (GFP), or a biologically active protein detectableby antibodies or enzymatic assay, to provide a means for identifyingbiologically stressed tissue in a transgenic animal. A heterogenousnucleic acid can further include antisense polynucleotides and dominantnegative encoding polynucleotides, which may be expressed in atransgenic non-human animal.

The term “transgenic” as used herein additionally includes any organismwhose genome has been altered by in vitro manipulation of the earlyembryo or fertilized egg or by any transgenic technology to induce aspecific gene knockout. The term “gene knockout” as used herein, refersto the targeted disruption of a gene in vivo with complete loss offunction that has been achieved by any transgenic technology familiar tothose in the art. In one embodiment, transgenic animals having geneknockouts are those in which the target gene has been renderednonfunctional by expression of antisense nucleic acid. As used herein,the term “transgenic” includes any transgenic technology familiar tothose in the art which can produce an organism carrying an introducedtransgene or one in which an endogenous gene has been renderednon-functional or “knocked out”.

The “non-human animals” of the invention include vertebrates such asrodents, non-human primates, sheep, dog, cow, pig, amphibians, andreptiles. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse. The “transgenicnon-human animals” of the invention are produced by introducing“transgenes” into the germline of the non-human animal. Embryonal targetcells at various developmental stages can be used to introducetransgenes. As previously noted, different methods are used depending onthe stage of development of the embryonal target cell.

A “transgenic” animal can be produced by cross-breeding two chimericanimals which include exogenous genetic material within cells used inreproduction. Various methods to make the transgenic animals of thesubject invention can be employed. Generally speaking, three suchmethods may be employed. In one such method, an embryo at the pronuclearstage (a “one cell embryo”) is harvested from a female and the transgeneis microinjected into the embryo, in which case the transgene will bechromosomally integrated into both the germ cells and somatic cells ofthe resulting mature animal. The use of a one cell embryo as a targetfor gene transfer has a major advantage in that in most cases theinjected DNA will be incorporated into the host gene before the firstcleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442,1985). As a consequence, all cells of the transgenic non-human animalwill carry the incorporated transgene. This will in general also bereflected in the efficient transmission of the transgene to offspring ofthe founder since 50% of the germ cells will harbor the transgene.Microinjection of such an embryo is the preferred method forincorporating transgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retro viral infection (Jaenich, R., Proc. Natl. Acad. Sci.USA 73:1260-1264, 1976). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The viral vector systemused to introduce the transgene is typically a replication-defectiveretro virus carrying the transgene (Jahner, et al., Proc. Natl. Acad.Sci. USA 82:6927-6931, 1985; Van der Putten, et al., Proc. Natl. Acad.Sci. USA 82:6148-6152, 1985). Transfection is easily and efficientlyobtained by culturing the blastomeres on a monolayer of virus-producingcells (Van der Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (D. Jahner etal., Nature 298:623-628, 1982). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellsthat formed the transgenic nonhuman animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retro viral infectionof the midgestation embryo (D. Jahner et al., supra). Methods to maketransgenic animals described generally above are described in U.S. Pat.No. 5,162,215, incorporated herein by reference.

In another such method, embryonic stem cells are isolated and thetransgene incorporated therein by electroporation, plasmid transfectionor microinjection, followed by reintroduction of the stem cells into theembryo where they colonize and contribute to the germ line. Methods formicroinjection of mammalian species is described in U.S. Pat. No.4,873,191, incorporated herein by reference. ES cells are obtained frompre-implantation embryos cultured in vitro and fused with embryos (M. J.Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al., Nature 309:255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci. USA 83:9065-9069, 1986; and Robertson et al., Nature 322:445-448, 1986).Transgenes can be efficiently introduced into the ES cells by asdescribed above. Such transformed ES cells can thereafter be combinedwith blastocysts from a nonhuman animal.

The analysis of expression of a transgene is essential in determiningthe utility of the transgenic animal produced. As with integrationanalysis, the presence or absence of similar or identical endogenouscounterparts will determine, to a degree, the strategies that may bemost useful. For transgenes that are unique (no endogenous counterpart)or contain some unique sequences, the strategies that can be used aremore straightforward. The presence of a novel RNA transcript or a uniqueprotein (or enzyme activity) is more easily determined than it is whenthe transcript or protein products are very similar to endogeneoustranscripts or proteins. As with integration analysis, molecular “tags”are also sometimes useful in that the transcripts will contain someunique identifying sequence that can be readily and unequivocallydetermined.

A nucleic acid construct of the invention can comprise a suitabledetectable marker expressed under the control of a stress-responsiveregulatory sequence that is active in a targeted, biologically stressed,tissue. “Detectable marker”, as used herein, refers to any identifiablecomposition useful for distinguishing cells containing a nucleic acidconstruct of the present invention from those cells that do not containsuch a construct.

It is also envisioned that biologically stressed tissue of a transgenicanimal of the invention can be identified by the presence of abiologically active protein product encoded by the construct of theinvention. Thus, a detectable marker of the invention also includesbiologically active protein products. The term “biologically activeprotein product”, as used herein, refers to products produced orsynthesized by a host cell as a result of the insertion of a transgeneinto the cell. In the present example, the cell can be part of atransgenic animal. In addition, the term encompasses those biologicalproducts that are secondary products of the activity encoded by atransgene.

In those cases where it is desirable for the detectable marker to encodea biologically active protein product, it is envisioned that antibodiescan be used to detect the presence of an antigenic determinant resultingfrom expression of the protein encoded by the heterologous DNA sequence.Such antibodies may, for example, recognize a specific epitope unique tothe expressed protein. The term “epitope”, as used herein, refers to anantigenic determinant on an antigen, such as a protein encoded by theheterologous nucleic acid, to which the paratope of an antibody, such asa protein encoded by the heterologous nucleic acid, binds. Antigenicdeterminants usually consist of chemically active surface groupings ofmolecules, such as amino acids or sugar side chains, and can havespecific three-dimensional structural characteristics, as well asspecific charge characteristics.

An antibody suitable for binding to a protein encoded by theheterologous nucleic acid is specific for at least one portion of anextracellular region of the protein encoded by the heterologous nucleicacid polypeptide. For example, one of skill in the art can use thepeptides to generate appropriate antibodies of the invention. Antibodiesof the invention include polyclonal antibodies, monoclonal antibodies,and fragments of polyclonal and monoclonal antibodies.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., Production of PolyclonalAntisera, in Immunochemical Protocols (Manson, ed.), pages 1-5 (HumanaPress 1992); Coligan et al., Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology,section 2.4.1 (1992), which are hereby incorporated by reference. Thepreparation of monoclonal antibodies likewise is conventional. See, forexample, Kohler & Milstein, Nature 256:495 (1975); Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A LaboratoryManual, page 726 (Cold Spring Harbor Pub. 1988), which are herebyincorporated by reference.

In addition, a detectable marker of the invention can be, for example, avisually detectable marker. In one embodiment, the invention utilizes avisually detectable marker protein that fluoresces directly uponillumination with light of an appropriate wavelength. Any fluorescentprotein can be used in the invention, including proteins that fluorescedue to intramolecular rearrangements or the addition of cofactors thatpromote fluorescence. For example, green fluorescent proteins ofcnidarians, which act as their energy-transfer acceptors inbioluminescence, are suitable fluorescent proteins for use in thefluorescent indicators. A green fluorescent protein (“GFP”) is a proteinthat emits green light, and a blue fluorescent protein (“BFP”) is aprotein that emits blue light. GFPs have been isolated from the PacificNorthwest jellyfish, Aequorea victoria, the sea pansy, Renillareniformis, and Phialidium gregarium. See, Ward, W. W., et al.,Photochem. Photobiol., 35:803, 1982); and Levine, L. D., et al., Comp.Biochem. Physiol., 72B:771982.

A variety of Aequorea-related GFPs having useful excitation and emissionspectra have been engineered by modifying the amino acid sequence of anaturally occurring GFP from Aequorea victoria. (See, Prasher, D. C., etal., Gene, 111:229, 1992); Heim, R., et al., Proc. Natl. Acad. Sci.,USA, 91:12501, 1994); U.S. Pat. Nos. 5,491,084; 5,625,048, incorporatedherein by reference). The cDNA of GFP can be concatenated with thoseencoding many other proteins; the resulting chimerics often arefluorescent and retain the biochemical features of the partner proteins.(See, Cubitt, A. B., et al., Trends Biochem. Sci. 20:448, 1995).Mutagenesis studies have produced may GFP mutants, some having shiftedwavelengths of excitation or emission. Such proteins are included in theinvention sensor.

A fluorescent protein is an “Aequorea-related fluorescent protein” ifany contiguous sequence of 150 amino acids of the fluorescent proteinhas at least 85% sequence identity with an amino acid sequence, eithercontiguous or non-contiguous, from the wild type Aequorea greenfluorescent protein. More preferably, a fluorescent protein is anAequorea-related fluorescent protein if any contiguous sequence of 200amino acids of the fluorescent protein has at least 95% sequenceidentity with an amino acid sequence, either contiguous ornon-contiguous, from the wild type Aequorea green fluorescent protein.Similarly, the fluorescent protein can be related to Renilla orPhialidium wild-type fluorescent proteins using the same standards.

Other selectable markers include DNA sequences encoding membrane boundpolypeptides. Such polypeptides are well known to those skilled in theart and contain a secretory sequence, an extracellular domain, atransmembrane domain and an intracellular domain. When expressed as apositive selection marker, such polypeptides associate with the targetcell membrane. Fluorescently labeled antibodies specific for theextracellular domain may then be used in a fluorescence activated cellsorter (FACS) to select for cells expressing the membrane boundpolypeptide.

It may also be useful in some circumstances to use a version of adetectable marker that is targeted to a specific subcellularcompartment. “Targeting signal sequence”, as used herein, refers to anynucleic acid or amino acid sequence useful for predetermining theintracellular or extracellular location of a molecule containing such asequence. Subcellular targeting of the detectable marker would beachieved by fusing the marker gene to a targeting sequence. For example,the nuclear localization signal from SV40 T antigen could be fused to,for example, a visually detectable marker such as GFP, which would leadto an accumulation of GFP in the nucleus. Numerous subcellular targetingsequences are known in the art. Using this well-known method, GFP hasbeen targeted to subcellular locations including the nucleus, themitochondria, the cell membrane, nuclear pores, the actin cytoskeleton,the golgi apparatus, transport vescicles and other locations.

The use of a targeting signal sequence is advantageous for threereasons. First, concentration of the detectable marker in a smaller areawithin the cell gives a brighter, more easily visualized fluorescentsignal. Second, some cells tend to exhibit a basal level of backgroundautofluorescence, which is generally distributed throughout thecytoplasm. Targeting GFP, for example, to a specific subcellularlocation permits the fluorescent signal generated by the transgene to bemore easily distinguished from background autofluorescence. A thirdadvantage of using subcellular targeting is that it allows sequentialintegration of different vector DNAs. This might be desirable insituations when it is desirable to have more than one gene expressed ina transgenic animal. For example, it might be desirable to express aspecific monoclonal antibody in a transgenic animal. In this case, afirst vector DNA expressing the heavy chain of a desirable antibodycould be integrated using GFP fused to an actin cytoskeletal targetingsequence. A second vector DNA carrying a GFP fusion to a nuclearlocalization signal and a light chain expression cassette could then beintegrated. Another example of when this technique might be useful is inthe case of proteins that must be processed by a particular protease inorder to attain their mature, active forms.

Materials and Methods

Cell Culture Conditions. The tumor cell line B/C10ME was cultured inhigh glucose Dulbecco's modified Eagle's medium (DMEM) containing 4.5mg/ml glucose supplemented with 10% fetal calf serum, 2 mM glutamine,and 1% penicillin-streptomycin-neomycin antibiotics. Transduced B/C10MEcells were maintained in 2 mg/ml of G418 respectively. To generateindividual transduced clones, transduced cells were plated into 96 wellplates by serial dilution, to a final concentration of 0.3 cell/well.Individual clones were then isolated and expanded.

Retroviral Vector Construction. The G1TkSvNa retroviral construct (Lyonset al., Cancer Gene Ther., 2:273, 1995) was obtained from GeneticTherapy Inc./Novartis (Summit, N.J.). G1NaGrpTk (FIG. 1) was constructedby removing the 356 by SV40 promoter region of a retroviral vectorG1NaSvTk (Hung et al., Int. J. Pediatr. Oncol., 4:317, 1997) by SalI andBglII and replaced with a 695 by rat grp78 promoter spanning -520 to+175 (Chang et al., Proc. Natl. Acad. Sci. USA, 84:684, 1987).Retroviral vector plasmid DNA was prepared by Qiagen Maxi Kit andtransfected into ecotropic retroviral producer cell line PE501. Theviral supernatant was harvested and an amphotropic retroviral producercell line PA317 was transduced and drug (G418) resistant clones wereselected. Retroviral vectors were collected and titered by NIH3T3 cells.

Western Blot. For the detection of HSVTK, GRP78 and β-actin, 20 mg ofcell lysate were prepared as previously described (Zhou et al., J. Natl.Cancer Inst., 90:381, 1998) and resolved on a denaturing sodium dodecylsulphate-8% polyacrylamide gel and transferred onto Hybondnitrocellulose membrane (Amersham Life Science Inc., Arlington Heights,Ill.). The membrane was blocked with 5% non-fat milk (Bio-RadLaboratories, Hercules, Calif.) in TBS buffer (20 mM Tris-HCl, pH 7.5,14 mM NaCl) for 1 h at room temperature prior to the incubation withpolyclonal rabbit anti-HSVtk antibody or monoclonal mouse anti-GRP78antibody (StressGen, British Columbia, Canada), or monoclonal mouseanti-b-actin antibody (Sigma Chemical Co.) 1 h at room temperature. Forall the primary antibodies, 1:1000 dilutions were used. The secondaryantibodies used were: goat anti-rabbit IgG conjugated with horseradishperoxidase (Promega, Wis.) and diluted 1:3000 in TBS buffer fordetecting HSVTK; and goat anti-mouse IgG conjugated with horseradishperoxidase (Promega, Wis.) and diluted 1:5000 in TBS buffer fordetecting GRP78 and β-actin. The immunocomplexes were detected with theEnhanced Chemiluminescence (ECL) kit (Amersham Life Science Inc.).

In Vitro GCV-sensitivity Assay. Individual clones of B/C10ME cellstransduced with either the G1TkSvNa or the GlNaGrpTk retroviral vectorwere seeded in duplicate at 5×10³ cells/well in a 6-well plate. On daythree after seeding, the cells were incubated with either control mediumor 0.1 mg/ml GCV. Fresh GCV was added daily to the cells, which werecounted every 3 days using the trypan blue dye exclusion method. Forglucose starvation treatment, on the second day after seeding, the cellswere maintained on glucose-free DMEM supplemented with dialyzed fetalcalf serum for a period of 30 h. After the 30 h, the cells wereincubated with 0.1 mg/ml GCV. GCV was added daily while the culturemedium was changed every third day for all cell cultures. When the cellsreached approximately 70% confluency, the cultures were transferred to10-cm diameter dishes.

Assay for In Vitro Bystander Effect. To measure the GCV killing effect,non-transduced B/C10ME cells were co-cultured with different ratio ofB/C10ME clonal cell lines stably transfected with GlNaGrpTk. Typically,a total of 3000 cells with various ratios (90%:10%; 75%:25%; 50%:50%)were plated in quadruplicate in 96 well plate and treated with 10 mg/mlGCV for 10 days. The number of remaining viable cells was measured bycell proliferation assay (Promega, Wis.).

Tumor Formation. Confluent cultures of B/C10ME clones were harvestedwith trypsin-EDTA (Gibco/BRL) and washed three times in PBS.Approximately 2×10⁷ viable cells were resuspended in 200 ml of PBS. Six-to eight-week-old BALB/c mice obtained from Jackson Laboratory weresubcutaneously injected with an 18-gauge needle in their right flank.Tumors were palpable within 12 days of inoculation and bi-perpendicularmeasurements were taken of the progressively growing tumor daily. Tumorgrowth was monitored by measurement of the larger and smaller diameters.At the indicated times post-injection, mice were injected with GCV dailyat a dosage of 100 mg/kg of body weight for about 10 days. Tumors werejudged to have regressed after losing both measurability andpalpability. For each retroviral construct, multiple injections of 2 to3 independently derived transduced clonal cell lines were performed.

Immunohistochemistry. Tumor tissues were removed, stored at −80° C. andcut by cryostat to 4 mm sections. The frozen sections were fixed by 10%formalin solution for 15 min and treated with 3% hydrogen peroxide. Arabbit polyclonal antibody against HSVtk was added to the sections for 1h at room temperature. After washing three times with PBS, a HRP labeledpolymer conjugated to goat anti-rabbit antibody (Dako, Carpenteria,Calif.) was added and incubated for 30 min. After three washes with PBS,the slides were stained with 3,3-diaminobenzidine (DAB) andcounterstained with methylgreen and covered with regular permount andviewed under a Zeiss microscope.

MicroPET Imaging of HSVtk Expression. The hypoxia-inducibility of theGRP78 promoter was demonstrated in vivo by microPET scanning to detectGRP78-driven HSV-tk gene expression, using the isotope-labeled substrate[¹⁸F] FHBG. Tumors were established by subcutaneous inoculation of TSAmurine breast cancer cells that had been stably pre-transduced with astandard replication-defective retrovirus containing the GRP78-drivenHSV-tk cassette. These tumors were then examined by microPET scanning inthe absence or presence of further hypoxia induction by photodynamictreatment. FIG. 6 shows microPET images of hypoxia inducible HSVtkexpression in a murine mammary adenocarcinoma model. The mcroPET scanwas performed as described (Gambhir et al. PNAS, 96:2333, 1999). Theisotope-labeled substrate was [¹⁸F]FHBG (Alauddin, Nuclear Medicine &Biology. 25:175, 1998). In Panel A, the grp78 promoter is able to drivehigh level HSVtk expression in sizable solid tumors. A tumor was formedin the left (L) shoulder area in a BALB/C mouse by injecting s.c. 2×10⁷of G1NaGRP-HSVtk transfected TSA cells. The microPET scan was performedwhen the tumor was about 1.5 cm in diameter. The red color denotes highHSVtk activity. In Panel B, the grp78 promoter is inducible by hypoxiaactivated by photodynamic treatment (PDT). Two tumors were formedsimultaneously on the left (L) and right (R) shoulder areas of a BALB/Cmouse the same as described in A. The microPET scan was performed whentumor sizes reached about 0.6 cm in diameter and about 12 hours afterthe tumor on the right had received PDT which induces hypoxia in vivo.The two tumors were of approximately the same size before PDT treatmentand the relatively larger contour seen on the image on the R tumor isdue to hemorrhage and edema after PDT treatment.

Production of Transgenic Animals. The present invention further providesa transgenic mouse line expressing the β-galactosidase (lacZ) genedriven by the GRP78 promoter. Significantly, lacZ staining was observedin a variety of tumor tissues that developed in the transgenic miceafter exposure to chemical carcinogens, but was not observed in anynormal organs. Furthermore, using a plasmid containing the grp78endoplasmic reticulum stress response element (ERSE) linked to theminimal MMTV promoter, extremely low basal levels and a 25-foldinduction by glucose starvation was observed upon transient transfectionin the human prostate cancer cell line PC3.

The transgene of the invention was injected into fertilized eggs fromsuperovaluated 4 to 5 week old F1 (C57BL/6J xCBA/J) females impregnatedby Fl (C57BL/6J×CBA/J) adult males. Psuedopregant females for embryotransfer were produced by matings between CD1 adult females andvasectomized CD1 adult males.

Mice of about 1.5 to 2 years of age were treated with the chemicalcarcinogen every week for 6 months. Tumors developed in both transgenicmice as well as non-transgenic controls. Tumors and normal organs wereexcised and stained for β-galactosidase expression. Blue color indicatesthat the grp78 promoter was active and driving the expression of theβ-gal gene. The results showed no expression in normal organs indicatinglow grp78 promoter activity in normal tissues but elevated expression intumorous and/or inflammatory tissues.

Results

Features of Stress-inducible grp78/BiP Promoter. Under glucosestarvation and anaerobic conditions, the grp78 promoter is highlyinduced. The mammalian grp78 promoter is functionally redundant andcontains multiple stress-inducible elements interacting with the CBF andYY1 transcription factors (Li et al., J. Biol. Chem., 268:12003, 1993;Roy et al., J. Biol. Chem., 271:28995, 1996; Li et al., Mol. Cell.Biol., 17:54, 1997). The genetic code for endoplasmic reticulum stresssignaling leading to grp gene induction consists of two units of a 19base pair (bp) sequence motif (CCAAT)N9(CCACG) (SEQ ID NO:1) termedERSE. This sequence contains a tripartite structure, with a highaffinity CBF/NF-Y binding site separated by precisely 9 by of a GC richsequence motif to a low affinity YY1 binding site.

In the construction of the retroviral vector GlNaGrpTk, the rat grp78promoter, spanning 520 by upstream and 175 by downstream of the site ofinitiation of transcription, serves as an internal promoter driving theexpression of the HSVtk gene (FIG. 1). This 695 by grp78 promotersubfragment contains three ERSEs, a TATA element and an internalribosome entry site, a unique and useful feature of the 5′ untranslatedregion of grp78 that allows internal initiation of translation (Macejaket al., Nature, 353:90, 1991). In the G1NaGrpTk vector, the MuLV LTRdirects the expression of the neo gene that is used as a selectionmarker. For comparison, instead of using a retroviral vector withanother internal promoter such as SV40 that has previously been shown tobe ineffective to drive a reporter gene in a tumor environment (Gazit etal., Cancer Res., 55:1660, 1995), the G1TkSvNa retroviral vector wasused. In this vector, the viral LTR drives the expression of the HSVtkgene, while the Simian virus SV40 promoter drives neo expression (FIG.1). The rationale for choosing G1TkSvNa is that it represents animproved retroviral vector for suicide gene therapy and is the vector ofchoice in current clinical protocols (Anderson, Nature, 392(Suppl):25,1998). Both vectors were transduced into B/C10ME, a murine fibrosarcomacell line that is syngeneic with the Balb/c mice. The advantage of theB/C10ME as a model system is that it has been previously establishedthat kinetics of tumor growth and subsequent regression can be readilymonitored in the recipient mice.

Glucose Deprivation Induces grp78-driven HSVtk Expression in vitro. Tocreate clonal B/C10ME cell lines with stably integrated retroviralvectors, the cells infected with the retroviruses were selected withG418. Serial dilution plating was performed after selection to isolateindividual clones. The individual clones were expanded and analyzed.Under standard culture conditions, B/C10ME cells transduced with eitherretroviral construct exhibited equivalent plating efficiencies andgrowth rates (see below). Thus, the basic growth properties of thetransduced cells in vitro were similar.

To test for the efficacy of the LTR and the grp78 promoter to driveexpression of the HSVTK protein, total cell lysates were prepared fromindividual clonal lines under normal culture and glucose starvedconditions. The proteins were separated by SDS-PAGE and subjected toWestern blot analysis. The levels of HSVTK, GRP78 and β-actin in eachsample was measured. As expected, there was no detectable HSVTK in thenon-transduced B/C10ME cells (FIG. 2). In the clonal line with the HSVtkgene driven by the LTR, there was HSVTK expression under normal cultureconditions. However, when the cells were subjected to glucose starvationfor 24 h, while the level of GRP78 was induced and the level of HSVTKwas reduced. In contrast, in the clonal cell lines with the HSVtk genedriven by the grp78 promoter, the level of HSVTK was upregulated inglucose-starved cells (FIG. 2).

To analyze HSVtk activity under normal and glucose-starved conditions,clonal cell lines derived from B/C10ME transduced cells with eachrespective retroviral construct were analyzed using an in vitroGCV-sensitivity assay (FIG. 3). For this purpose, about 5,000 cells wereseeded in duplicates in 6-well plates, and on the third day of seeding,either remained untreated, or incubated with 0.1 μg/ml of GCV. One setof cells was cultured in normal culture medium containing 4.5 mg/ml ofglucose, and an identical set of cells was maintained in glucose-freemedium supplemented with dialyzed fetal calf serum for 30 h prior to theaddition of GCV. Example of the GCV survival test for a typical B/C10MEderived clone transduced with G1TkSvNa (G1TkSvNa/clone #3) is shown inFIG. 3, Panel A. Without the addition of GCV the cells continued to growexponentially, and by the end of the 12th day, the cell number hadreached 4×10⁶. Addition of GCV resulted in loss of live cells at asimilar rate for both sets of cells. By the end of the 12th day, about1,000 cells survived (FIG. 3, Panel A). Thus, for the LTR-driven HSVtk,the sensitivity to GCV was similar in cells cultured in normal orglucose-free medium.

The results of the GCV survival assay for a typical clonal line(G1NaGrpTk/clone #3) derived from B/C10ME cells transduced withGlNaGrpTk is shown in FIG. 3, Panel B. Under normal culture conditions,the growth rate as well as sensitivity to GCV was similar to that drivenby the LTR. However, in contrast to the LTR-driven HSVtk cells, whenGlNaGrpTk transduced cells were pretreated with the glucose-free medium,decrease in viable cells was much more pronounced. Thus by day 9 therewere no more surviving cells. Further, to demonstrate these cellsexhibit a bystander effect, HSVTK-positive cells were co-cultured withvarious ratios of non-transduced HSVTK-negative cells. Over 90% killingwas observed when only 10% of GlNaGrpTk cells are present in the culture(FIG. 3, Panel C). Collectively, these in vitro studies show that theretroviral construct containing an internal grp78 promoter produceshigher levels of HSVtk inducible by glucose deprivation, therebyenhancing the sensitivity of tumor cells to GCV.

Complete Eradication of Tumors in G1NaGrpTk Transduced Cells. Todirectly compare the therapeutic efficacy of the G1NaGrpTk vector withG1TkSvNa, B/C10ME clones transduced with the respective retroviralconstructs were injected subcutaneously at a dose of 2×10⁷ cells perBALB/c mouse. As controls, the parental, non-transduced cells were alsoinjected. Tumors were palpable after 12 days of injection. At day 21,when the average tumor diameter reached about 2 cm, GCV wasadministered. The rationale for starting the GCV treatment when thetumor had reached a sizable mass instead of just being palpable is thatthis will offer a more vigorous test for the potency of the retroviralvectors. For the parental B/C10ME cells, upon addition of GCV, thetumors continued to grow at various rates and growth was arrested astumors reached substantial mass (FIG. 4, Panel A). In the nine miceinjected with three different G1TkSvNa clonal cell lines, the majorityof tumor growth was arrested upon GCV treatment for two to three daysbut subsequently, tumor growth continued (FIG. 4, Panel B). Thus, atthis stage of tumor growth, the LTR-driven HSVtk was insufficient tomediate efficient GCV toxicity. In contrast, in mice injected with theG1NaGrpTk clonal cell lines containing the internal grp78 promoterdriving HSVtk expression, tumor regression was observed in all four miceinjected with two independently derived clonal lines following GCVtreatment. By day 29, there were no visible tumors in any of the animals(FIG. 4, Panel C). Complete tumor eradication was also observed in mousemammary tumor clonal cell lines transduced with G1NaGrpTk. All miceremained healthy and developed no tumors after withdrawal of the GCVtreatment.

To confirm that higher efficacy of G1NaGrpTk is due to higher expressionof HSVTK within the tumor, immunohistochemistry-staining for the HSVTKprotein was performed with the tumor tissues. Examples of theimmunohistochemistry-staining using antibody against HSVTK in B/C10MEtumors are shown in FIG. 5. The parental cells showed the absence ofHSVTK protein staining (FIG. 5, Panel A). A much higher level ofstaining was detected in tumors derived from G1NaGrpTk transduced cells(FIG. 5, Panel C) as compared to that derived from G1TkSvNa (FIG. 5,Panel B). Notably, the HSVTK staining for the G1TkSvNa was in isolatedpatches, suggesting there were areas within the tumor unfavorable forLTR-driven gene expression. In contrast, the staining for G1NaGrpTk wasmuch more enhanced across the tumor section as previously observed withthe endogenous grp78 transcript and the neo mRNA driven by the grp78promoter. Thus, within the tumor environment, G1NaGrpTk containing aninternal stress-inducible grp78 promoter is more effective in directinghigh level HSVTK expression than the retroviral LTR.

In cancer gene therapy, a major technical difficulty is the lack ofspecificity in targeting suicide gene expression in the anatomic site oftumors. The present invention provides a novel approach to this problemby using a stress-inducible promoter from the grp78 gene to direct theexpression of the HSVtk gene in solid tumors. Increased grp78 proteinexpression is detected in chemical- and radiation-transformed cells, aswell as in tumor cells that become drug-resistant. Within the tumorenvironment, glucose deprivation, chronic anoxia, and acidic pH inducethe GRPs, in particular grp78. Thus, grp78 mRNA levels are elevated in avariety of tumors, correlating with tumor size. These results indicatethat in regions of the tumors deprived of glucose and oxygen, the cellsexperience a stress response resulting in the specific activation of thegrp78 promoter.

The present invention provides a truncated rat grp78 promoter with mostof the distal basal elements removed while retaining its array ofstress-inducible elements (FIG. 1). When used as an internal promoter ina retroviral construct, the truncated rat grp78 promoter can driveincreased expression of HSVTK in vitro under glucose-starved conditions(FIGS. 2 and 3). These in vitro studies confirm that the internal grp78promoter is capable of inducing a high level of marker gene transcriptin glucose-deprived cells, in contrast to the HaMSV LTR that wasrepressed. In vivo, the G1NaGrpTk retroviral vector was highly effectivein directing HSVTK expression within the tumor environment (FIG. 5),leading to complete eradication of sizable tumors in their syngeneichost after GCV treatment. The potency of G1NaGrpTk, coupled with theknown bystander effects of suicide gene approach, suggests that thistype of vector could offer distinct advantages in solid tumor cancertherapy.

In addition, the present study shows that mice with regressed tumorsremained tumor free after withdrawal of GCV treatment. These dataindicate that protective immunity might have been induced in such mice,preventing regrowth of tumors. In support, there are several examples oflong-lasting antitumor immunity in various tumor models in response toHSVtk transduction and GCV treatments. For example, the immune responseelicited by mammary adenocarcinoma cells transduced with interferon-γand suicide genes may induce regression of lung metastases (Nanni etal., Hum. Gene Ther., 9:217, 1998). These data indicate that tumorstransduced with suicide genes can be used as live anti-tumor vaccines(Santodonato et al., Gene Ther., 4:1246-, 1997). In support of the this,it has recently been discovered that induction of apoptosis in tumorcells leads to a dramatic change in antigen presentation which couldlead to enhancement of the cell mediated immune response to the tumor.

Transgenic animals containing a nucleic acid construct of the inventionwere produced and used to identify biologically stressed tissue in theanimal. FIG. 7 shows the presence of the LacZ transgene in transgenicmice. Panel A is a diagram of the grp78/LacZ Transgene constructcomprising about 3000 base pairs of the grp78 regulatory sequenceoperably linked to the LacZ gene. Panel B, upper gel, shows a Southernhybridization resulting in the identification of a LacZ nucleic acidsequence in transgenic animals (Tg 132-147) containing the constructshown in Panel A. In the lower gel, a grp78 cDNA probe that hybridizesto the grp78 gene was used to demonstrate that similar amounts of totalDNA were loaded in to each lane of the gel. The transgenic sequenceswere identified using a suitably labeled LacZ probe. Non-transgenic(Non-Tg) animals do not contain the LacZ sequence. Panel C is a bargraph showing the LacZ activity present in hamster cells transfectedwith a plasmid containing a nucleic acid construct shown in panel A(grp78/LacZ) or a plasmid expressing LacZ from the SV40 large T antigenpromoter sequence (SV40/LacZ). Cells were treated with the calciumionophore A23187 to induce biological stress. Untreated and treatedactivity is indicated.

Carcinogen treatment of wild-type (+/+), heterozygous for the grp78/LacZtransgene (Tg/+) or homozygous for the grp78/LacZ transgene (Tg/Tg). Thecarcinogen (7,12-dimethyl benz[a]anthracene) was applied subcutaneouslyon a weekly basis over a period of six months. Subsequently, normal andtumorous tissue were isolated and stained for detection of LacZexpression (FIG. 8).

Color photographs of normal (non-neoplastic) tissue derived fromtransgenic mice that are homozygous for the grp78/LacZ transgene (Tg/Tg)or tissue derived from wild-type (non-transgenic) mice (+/+) are sown inFIG. 9. The mice, and tissue derived therefrom, were treated asdescribed in FIG. 8.

Color photographs of tumorous tissues removed from mice treated asdescribed in FIG. 8 are shown in FIG. 10. Tissue from mice heterozygousfor the grp78/LacZ transgene (Tg/+), homozygous for the grp78/LacZtransgene (Tg/Tg) and wild-type (+/+) are indicated. Note that,following LacZ-specific histological staining, LacZ expression isindicated in tumorous tissue derived from Tg/+ mice as well as tissuederived from Tg/Tg mice.

Additionally, photographs of tumorous tissues removed from mice treatedas described in FIG. 8 are shown in FIG. 11. Tissue from miceheterozygous for the grp78/LacZ transgene (Tg/+) or homozygous for thegrp78/LacZ transgene (Tg/Tg) are indicated. Note that, followingLacZ-specific histological staining, LacZ expression is indicated intumorous tissue derived from Tg/+ mice as well as tissue derived fromTg/Tg mice.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-62. (canceled)
 63. A method for inhibiting cell proliferationassociated with glucose starvation comprising: a) transducing a targetcell capable of cell proliferation with a recombinant retroviral vectorcomprising a nucleic acid construct, the nucleic acid constructcomprising (i) at least one glucose responsive protein 78 (grp78)non-coding regulatory sequence comprising at least two endoplasmicreticulum stress elements (ERSE) as set forth in SEQ ID NO:1; and (ii) aheterologous nucleic acid sequence operatively linked to the regulatorysequence, wherein the heterologous sequence comprises a structural genethat encodes a biologically active protein that converts anon-therapeutically effective compound to a therapeutically-effectivecompound in vivo; b) activating the glucose responsive protein 78(grp78) non-coding regulatory sequence such that the heterologousnucleic acid sequence comprising a structural gene that encodes abiologically active protein is expressed; and c) contacting the cellwith a non-therapeutically effective compound that is subsequentlyconverted to a therapeutically-effective compound by the biologicallyactive protein, wherein the therapeutically effective compound inhibitscell proliferation associated with glucose starvation.
 64. A method forreducing a cell proliferative disorder associated with glucosestarvation in a subject comprising: a) locally administering to thesubject a recombinant retroviral vector comprising a nucleic acidconstruct, the nucleic acid construct comprising (i) at least oneglucose responsive protein 78 (grp78) non-coding regulatory sequencecomprising at least two endoplasmic reticulum stress elements (ERSE) asset forth in SEQ ID NO:1; and (ii) a heterologous nucleic acid sequenceoperatively linked to the regulatory sequence, wherein the heterologoussequence comprises a structural gene that encodes a biologically activeprotein that converts a non-therapeutically effective compound to atherapeutically-effective compound in vivo; b) transducing a target cellin the subject with a vector of a); c) activating the glucose responsiveprotein 78 (grp78) non-coding regulatory sequence such that theheterologous nucleic acid sequence comprising a structural gene thatencodes a biologically active protein is expressed; and d) contactingthe cell with a non-therapeutically effective compound that issubsequently converted to a therapeutically-effective compound by thebiologically active protein, wherein the therapeutically effectivecompound inhibits cell proliferation associated with glucose starvationthereby treating the cell proliferative disorder.
 65. The method ofclaim 64, wherein the subject is a mammal.
 66. The method of claim 65,wherein the mammal is a mouse.
 67. The method of claim 65, wherein themammal is a human.
 68. The method of claim 64, wherein theadministration is by in vivo administration.
 69. The method of claim 68,wherein the in vivo administration is by direct administration.
 70. Themethod of claim 64, wherein the cell proliferative disorder is aneoplastic disorder.
 71. The method of claim 70, wherein the neoplasticdisorder is selected from the group consisting of lung cancer,colon-rectum cancer, breast cancer, prostate cancer, urinary tractcancer, uterine cancer lymphoma, oral cancer, pancreatic cancer,leukemia, melanoma, stomach cancer, thyroid cancer, liver cancer, andbrain cancer and ovarian cancer.